Project Summary/Abstract Metabolism plays a fundamental role in cancer growth, diagnosis (e.g. FDG-PET), and treatment (e.g. antifolates, asparaginase). Over the past decade, research into cancer metabolism has flourished, contextualized by the realization that metabolic changes in cancer are triggered by oncogene signaling and accelerated by the emergence of new measurement tools. This NCI Research Specialist Award will study cancer metabolism using the most important modern tools: mass spectrometry and isotope tracers. Working together with Unit Director Joshua Rabinowitz and his lab, and a diverse set of collaborators from Rutgers Cancer Institute of New Jersey and other regional cancer centers (U Penn, MSKCC, NYU), I aim over the next 5 years to achieve the following: (1) Develop mass spectrometry-based analytical methods that enable more complete and accurate metabolome quantitation. I will focus in particular on developing analytical solutions for hard to measure metabolites of high relevance to cancer. These include unstable species such as redox cofactors (e.g., NADPH) and activated folates (e.g. 5,10-methylene-THF). (2) Apply these methods to measurement of the cancer metabolome, from cultured cells to human tumors. This will include studies on flash-frozen de-identified patient tumor specimens, from cancers where we already have a leadership position (pancreatic ductal adenocarcinoma, oncocytoma) and those that remain understudied for a metabolic perspective (thyroid, neuroblastoma). Building on our prior success in discovering the oncometabolite 2- hydroxyglutarate, a particular focus will be identifying unexpected or novel metabolites that are altered in cancer. (3) Combine these methods with isotope tracers to determine fluxes in cancer cells and in vivo tumors. We have a long-standing leadership position in developing isotope tracer methods for quantitating metabolic fluxes, including notable recent success in measuring NADPH production pathways using 2H-tracers. We have recently developed protocols for infusing a wide variety of 13C and 2H-tracers into mouse, with the goal of enabling quantitation of tumor metabolic flux. Collectively, these tracers cover central carbon metabolism, one- carbon metabolism, and methionine and glutathione metabolism. We will apply them to study metabolism in genetically engineered mouse models including of pancreatic cancer with and without Myc activation and lung cancer with and without autophagy deletion. Resulting data will provide critical insights into the actual metabolic pathophysiology of cancer in the native tumor microenvironment and will thereby inform treatment selection strategies and the development of novel therapeutics.